Techniques for obtaining microorganisms that can efficiently produce useful compounds, such as organic acids, amino acids, nucleic acids, or vitamins, through, for example, introduction of auxotrophic mutation or metabotropic mutation or the recombinant DNA technology have been developed. As shown in FIG. 1, a method for obtaining auxotrophs in which activity of Enzyme X for converting Compound P into Metabolite M has been deleted had been often employed in order to efficiently produce Compound P in a biosynthetic pathway or a metabolic pathway in which Metabolite M indispensable for the growth is generated from a carbon source through Compound P as an intermediate metabolite. When Compound P of interest is to be produced with such mutant, however, the addition of Metabolite M or the final product generated from Metabolite M to a medium was necessary, and the production cost was disadvantageously increased.
For example, it has been reported that shikimic acid as an intermediate metabolite in the shikimic acid pathway shown in FIG. 2 can be efficiently produced with the use of Escherichia coli in which shikimate kinase activity had been deleted (Patent Document 1 and Non-Patent Document 1). When the microorganisms were used, however, parahydroxybenzoic acid auxotrophy, p-aminobenzoic acid auxotrophy, and 2,3-dihydroxybenzoic acid auxotrophy were developed in addition to tryptophan auxotrophy, tyrosine auxotrophy, and phenylalanine auxotrophy. This necessitated the addition of these 6 types of compounds to the medium. While it has been reported that 3-dehydroshikimic acid as an intermediate metabolite in the shikimic acid pathway could be efficiently produced with the use of Escherichia coli in which shikimate dehydrogenase activity had been deleted, disadvantageously, such microorganisms would also require the above 6 types of compounds (Patent Document 2 and Non-Patent Document 2).
Through the introduction of mutations by means of, for example, treatment with mutagens, such as N-methyl-N′-nitro-N-nitrosoguanidine or ethyl methanesulfonate, UV or radiation application, or spontaneous mutation, a mutation for decreasing the amount of Enzyme X that converts Compound P into Metabolite M may be introduced so as to decrease the amount of Metabolite M produced. Thus, the amount of Compound P may be increased. In such a case, activity of Enzyme X remains, and a part of Compound P is constantly converted into Metabolite M as a consequence. Accordingly, conditions for maximizing the amount of Compound P produced are not easily determined.
The present invention discloses a method for efficiently producing Compound P without the addition of Metabolite M or the final product generated from Metabolite M to a medium by allowing a repressor to artificially direct the transcription of the gene x encoding Enzyme X, so as to regulate the amount of Metabolite M produced. However, no case has been reported in which the productivity of Compound P had been improved by regulating the transcription of the gene x with the use of another promoter, the transcription therefrom is repressed by a repressor, instead of the promoter native to the gene x.
When producing useful compounds with the use of microorganisms, useful compounds are often produced through a pathway branched from Compound P in a biosynthetic pathway or a metabolic pathway in which Metabolite M indispensable for the growth is generated from a carbon source through Compound P as an intermediate metabolite. In such a case, a method for obtaining auxotrophs in which Enzyme X activity had been deleted had been often employed. Such technique, however, required the addition of Metabolite M or the final product generated from Metabolite M to a medium, and the production cost would be disadvantageously increased.
Concerning the production of target substances with the use of the shikimic acid pathway having many branched pathways (FIG. 2), for example, many tryptophan-producing strains are known to have tyrosine and phenylalanine auxotrophy so as to prevent chorismic acid, as an intermediate metabolite, from entering into a tyrosine or phenylalanine synthetic pathway (Patent Document 3 and Non-Patent Documents 3 and 4). Also, phenylalanine-producing strains are known to have tyrosine auxotrophy (Patent Documents 4 and 5 and Non-Patent Document 5). When producing an aromatic amino acid of interest, accordingly, it is necessary to add another type of aromatic amino acid to a medium.
It is also reported that about 40 g/L protocatechuic acid can be produced by creating a mutant in which an enzyme that converts 3-dehydroshikimic acid (hereafter abbreviated to as “DHS”) into shikimic acid (i.e., Metabolite M) has been deleted and the amount of 3-deoxy-D-arabino-heptulosonate-7-phosphate (hereafter abbreviated to as “DAHP”) as a starting material in the shikimic acid pathway has been increased (Non-Patent Document 6). When such mutant is used, however, in addition to tryptophan auxotrophy, tyrosine auxotrophy, and phenylalanine auxotrophy, p-hydroxybenzoic acid auxotrophy, p-aminobenzoic acid auxotrophy, and 2,3-dihydroxybenzoic acid auxotrophy are developed. Accordingly, the addition of these 6 types of compounds to a medium would be required, disadvantageously. In addition, protocatechuic acid-producing strains have been attained by introducing a mutation into bacteria of Corynebacterium (Patent Document 6) or Brevibacterium (Patent Documents 7 and 8). Such strain requires nutritional sources, such as tyrosine, in order to produce protocatechuic acid. Further, the maximal amount of protocatechuic acid produced by such mutant is about 13 g/L, and such amount is less than the amount produced by an aromatic amino acid auxotroph (Patent Document 8).